It is now becoming possible to introduce into some of the major crop plants foreign genes of potential interest in those plants. This process of genetic engineering has been accomplished with certain model species, such as tobacco, petunia, and carrot, and has now been accomplished in such major crops species soybean and cotton. In procedures for the genetic engineering of plants, it is desired that the genetic transformation of the plant tissues be of the germ line of the plant tissues. Germ line refers to the inheritable genetic material of the plant which is permanently altered by the transformation process in such a fashion that the plant will pass to its progeny by normal genetic inheritance the inserted foreign gene. While the transformation of somatic, or non-germ line cells, may be desired in some instances, in general in the genetic engineering of crop plants it is desired that germ line transformation of plant lines be achieved as quickly and efficiently as possible.
The most common previously utilized technique for the genetic engineering of plants involves the use of the soil-dwelling plant pathogenic bacterium Agrobacterium tumefaciens. A. tumefaciens has the natural ability to transfer a portion of its DNA, referred to as T-DNA, into the genome of susceptible plant cells. By changing the native T-DNA in an Agrobacterium strain, it is possible to use this unique trait of Agrobacterium to transfer desired genes into single plant cells. If the introduced gene includes a selectable marker, such as a herbicide or antibiotic resistance trait, it is possible thereafter to select for transformed cells in a tissue culture by imposing the putative transformant tissues to the selection pressure of the appropriate antibiotic or herbicide. Unfortunately, in soybean almost all cultivars are resistant to Agrobacterium infection and are thus very resistant to transformation with Agrobacterium. In addition, antibiotic resistant markers, such as kanamycin resistance commonly used in Agrobacterium plant transformation procedures with other plant species, have been found to be of limited utility in soybean transformation experiments. Accordingly, while it may be possible to utilize Agrobacterium-mediated transformation techniques in soybean, it is a difficult endeavor because of the lack of effective selectable markers.
Other techniques for transforming plants do exist, however. In particular, there exists a general approach to the transformation of plant cells which is based on delivering the transforming DNA into the plant cells by coating the DNA onto small inert carrier particles which are physically hurled into the target plant tissues. The technique of particle-mediated plant cell transformation was first demonstrated with somatic cells in such tissues as the epidermal tissue of onion and other such model cell cultures. Klein et al., Nature, 327:70-73 (1987). Later the originators of the particle-mediated transformation technique were able to achieve genetically engineered tobacco plants by the transformation of tobacco in tissue culture, using a selectable marker, which was then subsequently regenerated into whole plants. Klein et al., Proc. Natl. Acad. Sci. USA, 85:8502-8505 (1988). Rather than attempting to use particle-mediated transformation techniques on plant cells in culture, another approach was developed in which the growing meristems of plants were subjected to a particle-mediated transformation event. From such a technique, stable transformation of the germ line of soybean plants was achieved. McCabe et al., Bio/Technology, 6:923-926 (1988). This technique is not dependent on the availability of a selectable marker for the plant species.
In developing the technique for the germ line transformation of soybean plants using a particle-mediated technique based on meristem transformation, it was discovered that the transformation events often resulted in chimeric plants, which are plants in which some, but not all, of the tissues had been genetically transformed with the introduced DNA. McCabe et al., supra. Although the technique was thus useful to create genetically engineered plants, it was somewhat burdensome in the sense that large numbers of tissues had to be subject to transformation events, and large numbers of plants had to be cultivated from the putative transformed tissues in order to discover those particular shoots and plants which properly expressed the introduced DNA. Therefore, the ability to identify transformation events early in the process giving rise to heritable germ line transformation of tissues creates the ability to effectuate dramatic savings in the practical and cost-effective genetic transformation of plants resulting in reduced labor costs and reduced time and energy expended in cultivating the non-transformed tissues which were subject to the transformation events.
In seeking a germ line transformation of a plant species, such as soybean, it would be helpful if the progenitor tissue of the germ cells of the plant were identified in the growing meristem or shoot. Unfortunately, the science of developmental morphology of plant cells has not developed to the point that the ancestor cells of the soybean germ cells is known. Accordingly, if it is a plant meristem or embryo that is being transformed, no present knowledge exists as to which precise cells in that meristem or embryo must be transformed to achieve germ line transformation. Therefore, any correlation between categories of cells transformed in a growing soybean plant and a germ line transformation event would have to be determined empirically.